Oriental freshwater mussels arose in East Gondwana and arrived to Asia on the Indian Plate and Burma Terrane

Freshwater mussels cannot spread through oceanic barriers and represent a suitable model to test the continental drift patterns. Here, we reconstruct the diversification of Oriental freshwater mussels (Unionidae) and revise their taxonomy. We show that the Indian Subcontinent harbors a rather taxonomically poor fauna, containing 25 freshwater mussel species from one subfamily (Parreysiinae). This subfamily most likely originated in East Gondwana in the Jurassic and its representatives arrived to Asia on two Gondwanan fragments (Indian Plate and Burma Terrane). We propose that the Burma Terrane was connected with the Indian Plate through the Greater India up to the terminal Cretaceous. Later on, during the entire Paleogene epoch, these blocks have served as isolated evolutionary hotspots for freshwater mussels. The Burma Terrane collided with mainland Asia in the Late Eocene, leading to the origin of the Mekong’s Indochinellini radiation. Our findings indicate that the Burma Terrane had played a major role as a Gondwanan “biotic ferry” alongside with the Indian Plate.


Results
Freshwater mussel fauna of the Indian Subcontinent. Here, we present the most comprehensive phylogenetic and distribution datasets on freshwater mussels (Unionidae) from the Indian Subcontinent sampled to date with supplement of related taxa from Indochina and Africa ( Fig. 2 and Supplementary Fig. 1). The phylogeny was reconstructed using partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI), small ribosomal RNA (16S rRNA), and the nuclear large ribosomal RNA (28S rRNA) genes (Dataset 1). Based on the multi-locus phylogeny, DNA-based species delimitation procedures ( Supplementary Fig. 2), and morphological data, we show that the Unionidae fauna of the Indian Subcontinent contains members of one subfamily, the Parreysiinae (Table 1). The total species richness of freshwater mussels on the Indian Subcontinent is rather uncertain due to the lack of DNA sequence data for multiple nominal taxa (Table 1). In summary, we propose a list of 25 valid species, almost all of which seem to be endemic to the subcontinent, though only 17 (68.0%) of those taxa were checked by means of a DNA-based approach. The 25 species recorded from the Indian Subcontinent belong to three tribes: Indochinellini (genus Indonaia Prashad, 1918: 8 species), Lamellidentini (genera Lamellidens Simpson, 1900 and Arcidopsis Simpson, 1900: 9 and 1 species, respectively), and Parreysiini (genus Parreysia Conrad, 1853: 6 species) (Figs. 3, 4 and 5)). One more genus, the monotypic Balwantia Prashad, 1919 (Fig. 5h), is considered here as Parreysiinae incertae sedis. The genera Arcidopsis, Balwantia, and Parreysia are endemic to the Indian Subcontinent, while each of the Indonaia and Lamellidens also contains three species from Western Indochina. Furthermore, there is one cementing bivalve species, Pseudomulleria dalyi (Smith, 1898) (Etheriidae), known to occur in India whose systematic assignment is still unclear (see "Discussion").
Macroevolution and evolutionary biogeography of the Parreysiinae. Our combined supercontinent-based biogeographic modeling (S-DIVA + DIVALIKE) reveals that this subfamily most likely originated and diversified on Gondwana and its fragments (probability = 1.00), with a secondary radiation of the so-called Mekong's Indochinellini (sensu   51 , a compact but diverse monophyletic subclade, in the Sundaland Subregion (probability = 1.00) (Fig. 2). The earliest split within the subfamily was associated with the separation of the Lamellidentini from other taxa by a dispersal event (probability = 1.00) and did occur in the Early Cretaceous (mean age = 135 Myr, 95% HPD = 125-144 Myr) ( Fig. 2: Event I). Our combined tectonic plate-based ancestral area reconstruction (S-DIVA + DIVALIKE) suggests that this split occurred on the Burma Terrane, Indian Plate or on both of these blocks with equal probability (Fig. 2). The Parreysiini + Leoparreysiini clade most likely separated from the Coelaturini + Indochinellini clade by a dispersal event (probability = 1.00) in the mid-Cretaceous (mean age = 111 Myr, 95% HPD = 103-119 Myr) ( Fig. 2: Event II). The Burma Terrane and Indian Plate are returned as the most probable ancestral areas during this event by our combined model (Fig. 2).  [38][39][40] . The Mogok-Mandalay-Mergui Belt 40 is placed here within the boundary of the Burma Terrane. The map was created using ESRI ArcGIS 10 software (https:// www. esri. com/ arcgis). The topographic base of the map was created with Natural Earth Free Vector and Raster Map Data (https:// www. natur alear thdata. com) and Global Self-consistent Hierarchical High-resolution Geography (https:// www. soest. hawaii. edu/ wessel/ gshhg). (Map: Mikhail Yu. Gofarov).  (Fig. 2). These vicariance events reflect direct connections (river captures) between freshwater systems of these terranes based on our combined biogeographic reconstruction (probability = 1.00 in almost all cases).

Taxonomy of freshwater mussels from the Indian Subcontinent
This taxonomic section is largely based on our novel phylogenetic and morphological research ( Fig. 2 61,62 , and Bhutan 63 to the Salween River in Myanmar 56 . Comments: This genus contains not less than 11 recent species, eight of which occur on the Indian Subcontinent and three in the Western Indochina (Table 1 and Fig. 4b-h). Here, we tentatively delineate these taxa to three informal species groups ( Fig. 2 and Table 1). The caerulea-group contains eight Radiatula-like species having an ovate or elongated shell of moderate thickness. The cylindrica-group joins two Parreysia-like species with a thicker, ovate shell. Finally, the involuta-group combines two peculiar species, sharing a thin, fragile . Events I-VII indicate a series of key biogeographic events, shaping the recent distribution of the subfamily (see "Results"). Nodal circle charts indicate the probabilities of certain ancestral areas based on the combined "tectonic plates" scenario (S-DIVA + DIVALIKE). The Sunda Plate contains the Indochina Block and Sibumasu Terrane 39 . Black color indicates an unexplained origin. Color symbols GW (Gondwana) and LR (Laurasia) indicate the results of the combined "supercontinents" scenario (S-DIVA + DIVALIKE) with the probabilities (P) of each ancestral area being given in square brackets. Stars at branches indicate reliable fossil record of the Mesozoic Parreysiinae in Africa (red) and India (yellow) with available fossil taxa being listed in the corresponding callouts. Taxonomic information on the Mesozoic fossil species from the Indian Subcontinent is given in Table 2    Comments: This genus contains 12 recent species, nine of which occur on the Indian Subcontinent and three in the Western Indochina (Table 1 and Fig. 3a-h). In this study, we provisionally delineate these species to two  Table 1).
The corrianus-group contains four species usually having a more or less elongated shell, while the marginalisgroup joins species with somewhat ovate or rounded shell. Conversely, the shell outline itself cannot be used for diagnostic purposes even between the two species groups, as the shell shape of taxa in this genus is extremely variable, and multiple intermediate forms do occur, e.g. those in Lamellidens marginalis ( Fig. 3f-g).   79 to Lamellidens and propose L. friersoni (Simpson, 1914) comb. nov. Hence, Velunio Haas, 1919 syn. nov., a monotypic subgenus (section) 75 of the genus Physunio Simpson, 1900, established for this taxon, should be considered a synonym of Lamellidens.
The nominal species Unio groenlandicus Mörch, 1868 was introduced based on a description and figure of Schröter 80,81 . This taxon cannot be attributed to Schröter 81 , because this author named it as "die breite Mahler-Muschel aus Grönland", which is not a binomial name. Mörch stated that it "is Unio testudinarius, Spgl. (U. marginalis, Lam.), a common shell from Tranquebar and other places in British East Indies" 80 . However, we cannot link Schröter's figure (pl. 9, Fig. 1) 81 to a Lamellidens species due to the lack of pseudocardinal teeth. Hence, Unio groenlandicus is here considered a nomen dubium.
There are two older available names belonging to Lamellidens, i.e. Unio testudinarius Spengler, 1793 and U. truncatus Spengler, 1793 that were described from Tranquebar [Tharangambadi, 11.0292° N, 79.8494° E, Kaveri Basin, Tamil Nadu, India] 82 . Later, Haas redescribed these nominal taxa and illustrated the holotypes 83 . Based upon morphological examination, Haas considered Lamellidens testudinarius as the oldest available name for L. marginalis, and placed L. truncatus as a synonym of this species 83,84 . Furthermore, Haas synonymized the majority of nominal Lamellidens taxa under the name L. testudinarius 84 . However, this concept was largely ignored by subsequent researchers 1,2,57 . The assigment of these nominal taxa to certain species is not straghtforward. Morphologically, the holotype of Lamellidens testudinarius is an ovate shell 83 that could be something from the marginalis-group, e.g. L. marginalis, L. mainwaringi or L. jenkinsianus. In its turn, the holotype of Lamellidens truncatus represents a narrower, elongated shell 83 that looks either like L. corrianus or even the recently described www.nature.com/scientificreports/ L. unioides. Here, we prefer to consider these nominal species as taxa inquirenda but their identity will be clarified in the future based on molecular analyses of topotype samples from Tamil Nadu. The three earliest fossil members of this genus were described from the Late Cretaceous Intertrappean Beds of the Deccan Plateau in India, i.e. †Lamellidens carteri (Hislop, 1860), †L. deccanensis (J. Sowerby in Malcolmson, 1840) comb. nov., and †L. vredenburgi Prashad, 1921 ( Comments: This genus contains six recent species endemic to the Indian Subcontinent (Table 1 and Fig. 5a-g). Here, we delineate these species to three informal groups ( Fig. 2 and Table 1). The keralaensis-group contains Parreysia keralaensis sp. nov. only (Fig. 5a,b). This new species represents the most distant phylogenetic lineage within the genus (Fig. 2). The corrugata-group comprises four species that are phylogenetically and morphologically close to each other, representing a species complex (Table 1 and Fig. 5c-f). Our time-calibrated phylogeny indicates that the radiation within this group occurred during the Miocene (Fig. 2). Finally, the rajahensis-group contains a single species, Parreysia rajahensis (Lea, 1841). Although the DNA sequences of this species are not available, it probably represents a distant phylogenetic lineage due to a number of specific conchological features such as very thick, triangular shell and massive hinge plate (Fig. 5g).
The synonymy of Parreysia taxa is revised here ( The new species can be distinguished from other Parreysia taxa by having a prominent, massive, rounded umbo and a specific wave-like sculpture over the umbo or through the entire shell surface (Fig. 5a,b and Supplementary Fig. 3). Additionally, it represents the most distant phylogenetic lineage within the genus (Fig. 2).

Doubtful and uncertain freshwater mussel taxa linked to India
In this section, we present a morphology-based overview of several nominal taxa, which were described by Constantine S. Rafinesque 96 . Subsequent researchers largely ignored these taxa as "indeterminate Unionidae" and even as "the worthless fabrications of Rafinesque" because of very poor and incomplete descriptions 97,98 . In the body of available literature on the types of Unionidae described by  , any mention of the type series for his Indian taxa is absent. Furthermore, we were unable to locate the current whereabouts of these types neither in European museums nor in those in the USA (including the ANSP Malacology Collection database; http:// clade. ansp. org/ malac ology/ colle ctions). Perhaps, the type lots have been sold to a private collector(s), because in the introduction of that paper Rafinesque offered for sale all the type shells described there 96 . Hence, the types are probably lost. Therefore, our decisions and comments are based exclusively on the original descriptions. Taxa, the protologues of which lack diagnostic features for reliable taxonomic identification, are considered here as nomina dubia.
A complete reappraisal of Rafinesque's nominal taxa linked to India 96 is given in Supplementary Note 3, while a brief summary of our taxonomic decisions is presented here. Diplasma marginata Rafinesque, 1831 is considered a nomen dubium, because its type locality is uncertain (River Tennessee or Hindostan) and the identity is unclear. Three more nominal species cannot be identified with certainty based on the original descriptions and are also considered nomina dubia: Diplasma similis Rafinesque 84 . Based upon a morphological examination of the holotype, we found that it conchologically corresponds to Diplodon parallelopipedon (Lea, 1834) (Hyriidae: Hyriinae), a South American species, which is known to occur in the Paraná Basin and coastal drainages of Uruguay 1,2 . Hence, its type locality was given in error. The formal synonymy is proposed here as follows: Diplodon parallelopipedon [= Unio digitiformis Sowerby, 1868 syn. nov.].

Discussion
Taxonomic richness and endemism of Oriental freshwater mussels. The Indian Subcontinent houses a rather taxonomically poor fauna of the Unionidae, which contains 25 species belonging to three Gondwanan tribes (Indochinellini, Lamellidentini, and Parreysiini) and one subfamily, the Parreysiinae. All these species are endemic to the region, except for Lamellidens nongyangensis Preston, 1912, a local population of which was recorded in Lake of No Return (Nongyang Lake) near the boundary between India and Myanmar. Our novel results confirm the conclusion of Bolotov et al. 19 that the Unionidae faunas of the Indian and Western Indochina subregions share almost 100% level of endemism at the species level and that multiple records of Indian species in Myanmar 57,110-113 were based on erroneous identifications. Furthermore, the tribe Parreysiini and the genera Arcidopsis, Balwantia, and Parreysia are unknown beyond the Indian Subregion.
The taxonomic richness of the Unionidae fauna in Western Indochina is 2.5 times higher compared with that on the Indian Subcontinent, with more than 60 species, but it represents an amalgam of the original Gondwanan taxa (Indochinellini, Lamellidentini, and Leoparreysiini), and the Paleogene immigrants from the Sundaland (Contradentini and Pseudodontini) 19 (Smith, 1898) was considered a possible Gondwanan relict 117 . Traditionally, this enigmatic "freshwater oyster" was placed in the Etheriidae based on morphological criteria 57,84,110,118,119 but phylogenetic reconstructions using a single available COI sequence of this taxon (acc. No. AF231750) repeatedly indicated that it is a unionid species belonging to the Parreysiinae 120-122 . Its close affinities with the Unionidae were previously assumed based on anatomical surveys 118,123 . Conversely, Graf & Cummings considered this COI sequence as potentially problematic due to the discordance of its phylogenetic position with morphological data 104 and returned Pseudomulleria dalyi to the Etheriidae 1 . The latter concept of Pseudomulleria is accepted in the most recent global checklist of freshwater mussel taxa 2 . Although the COI sequence under discussion seems to be correct, we did not include this taxon to the current list of the Indian Parreysiinae, because a final solution on its family-level placement requires an expanded set of DNA sequences and needs further research efforts 2 .
We show that several highland areas of the Indian Subcontinent harbor endemic taxa of freshwater mussels with restricted ranges ( Table 1). As it was expected 117 Basin). Finally, Parreysia rajahensis seems to be endemic to the Narmada River 97 . Hence, these freshwater systems should be considered of highest priority areas for freshwater mussel conservation at the national and global levels.
It was assumed that the Western Ghats could have served as a refugium for the autochthonous Gondwanan fauna during the Deccan eruptions 126 . At first glance, our data on the taxonomic diversity and endemism of freshwater mussels agree with this hypothesis. This mountain range represents a major evolutionary hotspot for a plethora of taxa with possible Gondwanan affinities such as scorpions 126 , freshwater gastropods 127 , freshwater fish 128,129 , frogs [130][131][132][133] , and evergreen trees 134 .
The Andaman and Nicobar archipelagoes, being a union territory of India, are located on a separate tectonic platelet, which is confined to the western margin of the Sunda Plate 38 . These islands may therefore harbor a specific freshwater mussel assemblage that should be different from those on the Indian Subcontinent and Burma Terrane. A single nominal taxon, Alasmodonta nicobarica Mörch, 1872, was described on the basis of one shell from the Nicobar Islands 135 . Based upon the original description 135 , this shell is irregularly oval, convex, with irregular growth lines; dorsal margin slightly arched, anterior margin narrowed and rounded, ventral margin slightly concave, posterior margin narrowed and slightly prominent; shell color is olive, with darker bandages and numerous dark green rays; umbo is not prominent, eroded; pseudocardinal teeth are almost completely absent, lateral teeth are lamellar. Simpson placed this nominal taxon in the genus Pseudodon sensu lato 70 based on Mörch's comments in the protologue 135 but later transferred it to Pletholophus Simpson, 1900 with respect to the expert opinion of Haas, who has examined the holotype of Alasmodonta nicobarica 79 . Currently, it is considered a synonym of Pletholophus tenuis (Griffith & Pidgeon, 1833) (Unioninae: Cristariini), an East Asian species 2 . However, if its type locality is stated correctly, it cannot be assigned to Pletholophus tenuis from a biogeographical point of view 35 . At first glance, it may be a member of the genus Monodontina Conrad, 1853 (Gonideinae: Pseudodontini). This genus can be distinguished from other taxa by having an ovate or rounded shell and weakly developed pseudocardinal teeth 36 , which aligns with the original description of Alasmodonta nicobarica. The Monodontina taxa sometimes share green rays through the periostracum. The genus Monodontina is known to occur in southern Myanmar (Lenya River), southern Thailand, Chao Phraya and Mekong basins, and throughout Malaysia and the Greater Sunda Islands 23 , and, hence, could theoretically be found on the Nicobar Islands. Here, we propose Monodontina nicobarica (Mörch, 1872) comb. nov. as a preliminary taxonomic hypothesis that needs to be checked by means of a DNA-based approach. The Great Nicobar Island with its numerous rivers and streams flowing through primary tropical forests could indeed house some interesting freshwater mussel taxa and must be a focus of future collecting efforts. www.nature.com/scientificreports/ Here, we present an updated reconstruction of the origin and diversification of the Parreysiinae based on our novel biogeographic modeling, expanded paleontological data set, and the newest tectonic and paleomagnetic reconstructions [39][40][41][42]48,50,137 138 . These taxa were considered the earliest members of the modern crown-group of the Unionidae in Jurassic Africa 138 . In our opinion, these rare fossils could be linked to the MRCA or a steam group of the Parreysiinae based on the hinge structure. The initial breakup of East Gondwana from West Gondwana started approximately 160 Myr, separating the Indian Plate together with its proposed satellites from continental Africa 45,139 . Hence, the Parreysiinae MRCA most likely colonized India earlier (Fig. 6).

Gondwanaland origin and diversification of the
Our scenario further suggests that the earliest diversification in the subfamily occurred on an ancient landmass, containing the Indian Plate and Burma Terrane, which were joined through the Greater India, in the Early Cretaceous 47,139 (Fig. 7a). During that period, two large clades, i.e. Lamellidentini (Fig. 2: Event I) and Parreysiini + Leoparreysiini (Fig. 2: Event II) were separated. The splits, outlined above, coincided with a complete disappearance of unionids and margaritiferids in the Early Cretaceous deposits of continental Africa, probably reflecting a major extinction event 138 . Though it roughly coincides with the global Tithonian extinction event 138 , the post-Jurassic disappearance of naiads in Africa could also be linked to active development of a large system of rifts, leading to intercontinental marine transgressions [140][141][142] . Thereby we could assume that an ancient landmass, which consisted of the modern Indian Plate, Greater India, and Burma Terrane 39,40,42,48,137 , played a significant role as a refugium for freshwater mussels in the Early Cretaceous. Perhaps some geographic barriers on this landmass such as mountain ranges drove the early macroevolution of the Parreysiinae, as it was suggested for the diversification patterns in the Hyriidae 18 .
Our modeling reveals that a re-colonization event of the Parreysiinae from the Indo-Burma refugium into Africa most likely occurred in the mid-Cretaceous (Fig. 7b). This dispersal was followed by a vicariance event (mean age = 98 Myr) that lead to the origin of the African tribe Coelaturini (Fig. 2: Event III). A similar mean age was obtained for the split between the African Parachanna and Oriental Channa clades of snakehead fishes (Channidae) using a set of crown fossil calibrations 129 . The sister family Aenigmachannidae, a unique subterranean lineage from Western Ghats, separated from the Channidae approximately 110 Myr ago 129 . The pattern, outlined above, predicts a hypothetical land bridge between the Indian Plate and Africa nearly 100-110 Myr ago, probably through Madagascar 45,143 . Conversely, India could have reestablished biotic connections with Africa during its collision with the Kohistan-Ladakh Arc along the Indus Suture in the Late Cretaceous (ca. 85 Ma) 45 , although this geological event postdates our divergence age for the Coelaturini. Briggs assumed that India always remained close to Africa and Madagascar during its northward motion 144 . Our results, however, suggest that the Indian Plate was linked to Africa sometime in the mid-Cretaceous but this connection was lost afterwards.
Three subsequent splits in the subfamily Parreysiinae reflect the segregation of the Indian Plate and Burma Terrane during the Late Cretaceous (mean age interval 96 to 74 Myr; Fig. 2: Events IV, V, VI). There is no evidence of any connection between these landmasses during a nearly 50-Myr period (74 to 26 Myr), starting near the Campanian-Maastrichtian boundary and covering the entire Paleogene epoch (Fig. 7c). Our review of available fossils from the Deccan Intertrappean Beds reveals that members of Indonaia, Lamellidens, and Parreysia were presented on the Indian Plate 145 right before the Cretaceous -Paleogene (K-Pg) boundary 146 ( Table 2 and Supplementary Note 2). These paleontological findings support our ancestral area reconstruction indicating the Indian origin of these genera. Van Damme et al. noted that Deccan fossils could also belong to the Hyriidae because they often share a crenate or wavy ventral margin 138 . However, such "plicate" forms could independently evolve in different unionoid families, e.g. the Margaritiferidae (Margaritifera marrianae Johnson, 1983 147 and Pseudunio flabellatus (Goldfuss, 1837) 148 ) and Unionidae (genera Amblema Rafinesque, 1820 149 , Tritogonia Agassiz, 1852 150 , and others).
The Burma Terrane collided with the Sunda Plate in the Late Eocene (mean age = 38 Myr) that is reflected by the dispersal event of the Mekong's Indochinellini from the terrane to mainland Asia (Figs. 7d, 2: Event VII). During the same period, members of the tribes Contradentini and Pseudodontini, belonging to the subfamily Gonideinae, colonized the Burma Terrane from Asia (Sunda Plate) that leads to the endemic Pseudodon and Yaukthwa radiations in Western Indochina 23 . The Sundaland itself most likely represents an ancient evolutionary hotspot for the Gonideinae, because two endemic, deeply divergent tribes were recently discovered from Borneo, i.e. the Ctenodesmini Pfeiffer, Zieritz, Rahim & Lopes-Lima, 2021 and Schepmaniini Lopes-Lima, Pfeiffer & Zieritz, 2021 28 . Though these Bornean clades are yet to be involved into any time-calibrated phylogeny, their phylogenetic position (sister to the Contradentini + Rectidentini and to Pseudodontini, respectively) undoubtedly indicates a Late Mesozoic separation 28 .
A few species-level splits discovered in the genera Indonaia and Lamellidens indicate that the first re-connection of the Indian Plate and Burma Terrane did occur at the Oligocene -Miocene boundary (mean ages 26 to 24 Myr). Several additional faunal exchanges between these landmasses during the Miocene (mean ages 12 to 8 Myr), most likely reflecting river (stream) capture events, were also uncovered by our phylogeny. These range expansions could be traced in multiple fossil records of Indonaia, Lamellidens, and Parreysia species from Miocene deposits throughout Pakistan, India, Nepal, and Myanmar [65][66][67][68][69] . Perhaps, the exchanges between freshwater mussel faunas of the Indian Subcontinent and surrounding landmasses during the Miocene were triggered by humid and warm climatic episodes, as it was shown for freshwater gastropods 127 www.nature.com/scientificreports/ Interestingly, none of the unionid mussels seems to follow the so-called "Into India" scenario, though this pattern frequently occurs in Indian freshwater gastropods 127,153,154 , frogs 155,156 , and other animals. In contrast, our biogeographic models trace multiple "Into Burma" expansion events from India and Sundaland, starting since the Burma Terrane -Asia collision in the Late Eocene.
Burma Terrane as a second "biotic ferry" from Gondwana to Asia. There are multiple evidences that the Indian Plate has served as a "biotic ferry", transferring a derivative of the aboriginal Gondwanan biota to Asia 44,[157][158][159] . The iconic examples of taxa that are thought to have arrived to Asia by this way were discovered among caecilians 160,161 , frogs 132 , freshwater fishes 129,162 , freshwater crabs 163 , centipedes 164 , scorpions 126 , tarantulas 165 , and various plants 166,167 . Our study reveals that unionid mussels, a primarily freshwater group, the dispersal of which requires direct links between landmasses, should surely be added to the list of "passengers" that have travelled through the Tethys Ocean on this tectonic block. Furthermore, we show that the Burma Terrane could be considered a separate "biotic ferry" that also carried members of Gondwanan biota to Asia  . We assume that the MRCA of this subfamily migrated through freshwater systems of the African Plate and/or Arabia to an ancient landmass, containing the Indian Plate (with Greater India) and Burma Terrane. Red star indicates fossil records of the earliest African crown-group unionid mussels from Mid-to Late Jurassic deposits in Niger, i.e. †Coactunio iguallalensis, †Rostrunio lapparenti, and †Tuaregunio agadesensis 138 . IP Indian Plate, BT Burma Terrane, GI Greater India, SP Sunda Plate (with the Indochina Block and Sibumasu Terrane), SL Sri Lanka, MG Madagascar. Color filling is as follows: Burma Terrane (pink), Greater India (light orange), modern land (light yellow), proposed ancient land (light green), and ocean surface (light blue). The paleo-map was reconstructed using GPlates v. 2.3 (https:// www. gplat es. org) 205 and corresponding data sets [206][207][208][209] 205 and corresponding data sets [206][207][208][209] , with additional modifications according to a set of novel tectonic and paleomagnetic models [39][40][41][42]48,137,139  www.nature.com/scientificreports/ ( Fig. 7a-d). The high degree of endemism discovered in freshwater mussels on the Burma Terrane (and on the Indian Subcontinent as well) reveals that the Gondwanan "biotic ferries" have served as insular evolutionary hotspots, at least during the entire Paleogene (Fig. 7c). Our results support the hypothesis on insular endemism patterns (the so-called "endemic insularity syndrome") discovered in the paleo-biota from the mid-Cretaceous Burmese amber [168][169][170] . Earlier, it was suggested that several non-Indian Gondwanaland fragments such as the Burma and Lhasa terranes might have transferred Gondwanan lineages into Asia but any direct biogeographic evidence supporting this idea was not available 127 . The body of literature on this issue is still very limited, and a few available reports are based exclusively on paleontological data. First, a review of biogeographic affinities of numerous plant and animal taxa discovered in the mid-Cretaceous Burmese amber (ca. 100 Myr; near the Albian-Cenomanian boundary) reveals that this biota represents a selection of Gondwanan organisms and that the Burma Terrane could not have separated from East Gondwana before the Early Cretaceous 49 . From this perspective, ancestors of several secondary freshwater/estuarine and terrestrial groups of Mollusca discovered in Burmese amber such as †Palaeolignopholas piddocks (Pholadidae) 171 and some land snail taxa (Diplommatinidae and Pupinidae) 172,173 may have also arrived to Asia with the Burma Terrane. The discovery of a freshwater pond snail (Lymnaeidae) in this amber, however, could be linked to a long-distance dispersal event 174 . Perhaps it was not a transoceanic dispersal as such but an expansion from the nearby Indian Subcontinent, because the Deccan Trap sedimentary sequence harbors a diverse and species-rich assemblage of fossil freshwater snails, containing the Lymnaeidae, Planorbidae, Pomatiopsidae, Succinidae, Thiaridae, Valvatidae, and Viviparidae taxa 175 . Second, on the basis of a comprehensive survey on the Eocene flora of Myanmar, the Burma Terrane was considered a Gondwanan fragment that collided with Asia in the Late Eocene (ca. 41 Myr) and facilitated floristic exchange between the terrane, Indian Plate, and Asian mainland 176 . The dating of the Burma Terrane-Asia collision recovered in this research aligns with our estimate of 38 Myr inferred from the time-calibrated phylogeny of the Parreysiinae.

Conclusion
Our research presents the first DNA-based evidence that the Burma Terrane transferred an ancient derivative of Gondwanan biota to Asia, as India did. These results agree with a growing body of modern paleontological, tectonic, paleomagnetic, and geological research, supporting a Gondwanan origin of the Burma Terrane and its northward rafting through the Tethys Ocean [39][40][41]48,49,176 . Based on biogeographic patterns that were discovered in freshwater mussels (Unionidae: Parreysiinae), we propose that this terrane was a part of an ancient landmass, also containing the Indian Plate and Greater India, from the Middle Jurassic (ca. 160 Myr) to the terminal Cretaceous (ca. 75 Myr). Later on, during the Paleogene, the Burma Terrane was an isolated island that has collided with mainland Asia (Sunda Plate) in the Late Eocene (ca. 40 Myr). The biogeographic reconstruction presented here could be used as supplement to modern plate tectonic models, repeatedly indicating northward drifting of the Burma Terrane alongside the Indian Plate 40,177 . In general, our scenario of tectonic evolution of the region differs from other available scenarios 40 by the position of the Burma Terrane in relation to that of the Indian Plate and Greater India.
From this perspective, mainland Southeast Asia represents a Late Eocene collision zone of two tectonic blocks (Burma Terrane and Sunda Plate), initially housing completely different biotas 176 . These blocks could roughly be delineated via the Sagaing Fault and along the northern part of the Tenasserim Range through the Three Pagodas and Ranong faults 178 . This unique pattern was largely overlooked until recently which sometimes lead to incorrect conclusions on the origin and diversification of certain taxa, e.g. onychophorans 179 . To avoid possible reconstruction failures, Western Indochina should be coded as a separate, Gondwana-derived ancestral area in statistical biogeographic and paleontological models. Furthermore, the origin and role of several geographic barriers linked to the collision zone such as the Isthmus of Kra 23 and the Salween -Mekong drainage divide 35 must be re-considered based on these new findings.

Methods
Data collection. Freshwater mussel samples were collected from various localities in India, Nepal, and Myanmar from 2012 to 2020. A small foot or mantle tissue snip from each specimen from Myanmar and Nepal was fixed with 96% ethanol immediately after collection 19,22,23 . For the Indian samples, hemolymph was preferred as the source of genomic DNA. The hemolymph samples (0.2 ml per one specimen) were collected using a standard approach 180 , and genomic DNA was isolated from 0.1 ml of fresh hemolymph using the NucleoSpin® Tissue Kit (Macherey-Nagel GmbH & Co. KG, Germany), following the manufacturer protocol. The partial sequences of the mitochondrial COI, 16S rRNA, and the nuclear 28S rRNA genes were generated using standard protocols described in our earlier works 19,53,56 . The COI sequences of samples from Nepal were generated using the LCO1490 and COIschneck primers pair 181 , while those from Indian samples were obtained with the standard Folmer's primers 182 . Additional DNA sequences of Indian and African taxa were obtained from NCBI's GenBank (Datasets 1 and 2).
The dry shell vouchers and ethanol-preserved complete specimens collected by us were deposited in the following collections: FBRC ZSI-Freshwater Biology Regional Centre, www.nature.com/scientificreports/ Project (MUSSELp) Database (http:// mussel-proje ct. uwsp. edu) was used as a reliable source of taxonomic, bibliographic, and morphological information on nominal taxa of freshwater mussels 1,2,183 .

Phylogenetic analyses.
To reconstruct multi-locus phylogeny of the Parreysiinae (3 codons of COI + 16S rRNA + 28S rRNA), we compiled an alignment with 61 unique species-level haplotypes, including four outgroup taxa, that were selected from 203 sequenced specimens (Datasets 1 and 2). The maximum likelihood and Bayesian phylogenies were calculated using IQ-TREE v. 1.6.12 184 and MrBayes v. 3.2.7a 185 , respectively. The IQ-TREE 184 analysis was run using an automatic identification of the best evolutionary models for each partition 186 and an ultrafast bootstrap algorithm 187 via an online server (http:// iqtree. cibiv. univie. ac. at) 188 . The Bayesian analysis was performed through the CIPRES Science Gateway 189 . We assigned the best-fit evolutionary models to each partition based on the second-order Akaike information criterion (AICc) of MEGA7 190 as follows: GTR + G (1st codon of COI); TN93 + G + I (2nd codon of COI); HKY (3rd codon of COI); GTR + G + I (16S rRNA); and GTR + G (28S rRNA). The MrBayes settings were as follows: two runs (each with 50,000,000 generations), four MCMC chains (three cold and one heated; temp = 0.1), sampling every 1000th generation, and a 15% burn-in.
Species delimitation. Two species delimitation approaches were applied through available web-services, that is, the Poisson Tree Process (PTP) modeling (http:// mptp.h-its. org) 191 and ASAP (assemble species by automatic partitioning; https:// bioin fo. mnhn. fr/ abi/ public/ asap) 192 . As an input tree, we used the maximum likelihood COI phylogeny of the Parreysiinae inferred from IQ-TREE v. 1.6.12 184 . An initial alignment with 196 ingroup COI sequences was compiled (Dataset 1). This alignment was converted to 173 unique haplotypes using an online sequence toolbox, FaBox v. 1.5 (https:// birc. au. dk/ ~palle/ php/ fabox) 193 . Four Pseudodontini haplotypes were used as outgroup (Dataset 1). The IQ-TREE analysis was run through an online server (http:// iqtree. cibiv. univie. ac. at) 184 with settings as described above. Each species-level Molecular Operational Taxonomic Unit (MOTU), probably corresponding to a biological species, was checked with morphological criteria such as the shell shape, shell sculpture, umbo position, structure of pseudocardinal and lateral teeth, and shape of muscle attachment scars 56 . To link each MOTU to certain nominal species, the conchological features of available specimens were compared with the original taxonomic descriptions 23 .
Divergence time estimation. Divergence ages were estimated using BEAST v. 2.6.3 194,195 . The time-calibrated phylogeny was reconstructed based on an external COI evolutionary rate (1.5E-9 substitutions per site per year) 196 . This rate was obtained using a comprehensive set of mitochondrial genome sequences and several reliable fossil calibrations for Unionidae taxa 196 and largely agrees with earlier estimates of the COI molecular clock rate in freshwater mussels 147,197 . For BEAST runs, we used the same multi-locus dataset as for the IQ-TREE and MrBayes phylogenetic analyses (3 codons of COI + 16S rRNA + 28S rRNA). The molecular clock rate was assigned to the COI partition only. The HKY + G model was applied for each gene partition based on our earlier considerations 19 . We applied a strict clock algorithm with the Yule speciation process as the tree prior 198 . The BEAST runs were performed through the CIPRES Science Gateway 189 . Three independent BEAST runs were performed, each with 150,000,000 generations and tree sampling every 1000th cycle. The resulting log files were checked visually with Tracer v. 1.7.2 199 . The Effective Sample Size (ESS) values of all the parameters in the combined runs were found to be > 300 after a 50% burn-in. The final tree set was generated using LogCombiner v. 2.6. 6 194,194 with an additional re-sampling every 5,000th generation and an appropriate burn-in. The consensus tree was found with TreeAnnotator v. 2.6.6 194,194 . Ancestral area reconstruction. First, we reconstructed possible ancestral areas using data on the distri-  202 . As input files, we used the set of trees and the consensus phylogeny obtained from BEAST runs (see above). The branch length of the trees was converted from years to Myr using a "Scaling Branch Length" option of RASP v. 4.2 202 with an appropriate scaling coefficient (1.0E-6). The four outgroup taxa were removed from the trees using a "Remove Selected Groups" option of RASP v. 4.2 202 . The biogeographic analyses were run with default settings (max areas = 2) but without a set of + J models checking for founder-event speciation 200 , because these models appear to be rather doubtful from a statistical and conceptual point of view 203 .
To find the most appropriate biogeographic models, we conducted a comparative analysis of the relative probability of BioGeoBEARS models (DEC, DIVALIKE, and BAYAREALIKE) using the log-likelihood (LnL), AICc, and model weight according to ΔAICc (AICc wt) [200][201][202] . For the "supercontinents" reconstruction, the DIVALIKE model shared higher relative probability compared with others with respect to the AICc wt value (Supplementary Table 1). In its turn, the DEC model could be chosen according to that criterion among those reconstructing ancestral areas on the basis of tectonic plates (Supplementary Table 1 www.nature.com/scientificreports/ and S-DIVA 204 models using the "Combine Results" option of the software 202 . These combined models were used in subsequent biogeographic analyses and reconstructions. Tectonic plate modeling. The tectonic plate reconstructions for selected time intervals were calculated using GPlates 2.3 software (https:// www. gplat es. org) 205 and a corresponding set of digital layers on topological plate model [206][207][208][209] . Additional settings were obtained from a set of novel tectonic and paleomagnetic reconstructions [39][40][41][42]48,137,139 . The paleogeographic positions of the Burma Terrane and Greater India at 165-170, 135, 100, 75, and 40 Myr were modified manually on the basis of our time-calibrated phylogenetic reconstruction and statistical biogeographic models (see above).
Nomenclatural acts. The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature (ICZN), and hence the new name and combinations contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank (http:// zooba nk. org), the online registration system for the ICZN. The LSID for this publication is: http:// zooba nk. org/ urn: lsid: zooba nk. org: pub: FABE4 C0F-313E-4AB8-803F-35235 95D9A 39. The electronic edition of this paper was published in a journal with an ISSN, and has been archived and is available from PubMed Central.